Deciphering cis-regulatory logic with 100 million synthetic promoters

In order to generate data suitable to decipher cis-regulatory logic, we generated ~100 million synthetic promoters (in yeast) comprised of random DNA and measured their expression by FACS (sorting into 18 bins). Overall design: One such library (pTpA) was tested in glucose (YPD), galactose (YPGal), and glycerol (YPGly), the other (Abf1TATA) only in glucose. A separately constructed pTpA library of limited complexity was assayed separately, yielding higher quality expression measurements. A final library containing diverse promoter scaffolds was tested in glucose. The sequences flanking the random 80 bp oligo were as follows: the pTpA distal region was (pT) GCTAGCAGGAATGATGCAAAAGGTTCCCGATTCGAACTGCATTTTTTTCACATC and proximal region (pA) was GGTTACGGCTGTTTCTTAATTAAAAAAAGATAGAAAACATTAGGAGTGTAACACAAGACTTTCGGATCCTGAGCAGGCAAGATAAACGA (up to the theoretical TSS). For Abf1TATA, the distal region was GCTAGCTGATTATGGTAACTCTATCGGACTTGAGGGATCACATTTCACGCAGTATAGTTC and proximal was GGTTTATTGTTTATAAAAATTAGTTTAAACTGTTGTATATTTTTTCATCTAACGGAACAATAGTAGGTTACGCTAGTTTGGATCCTGAGCAGGCAAGATAAACGA. In both cases, 80 Ns were inserted in between proximal and distal regions. The scaffold library contained a variety of proximal and distal DNA fragments, and was grown in YPD. The Native80 and designed-sequence libraries were grown in YPD and used a strain of S288C with Ura3 deleted. All others used strain Y8205 (Boone Lab).